Sequential delivery of immunogenic molecules via adenovirus and adeno-associated virus-mediated administrations

ABSTRACT

An immunogenic regimen is provided. The regimen involves sequential administration of a recombinant adenoviral vector and a recombinant adeno-associated viral vector, each of which delivers a heterologous expression cassette encoding the same immunogenic product, or a cross-reactive immunogenic product. Also provided are products containing the vectors for use in the regimen of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a national stage application under 35 U.S.C. 371 ofPCT/US05/014556, filed Apr. 27, 2005 which claims the benefit under 35U.S.C. 119(e) of the priority of U.S. Patent Application No. 60/565,936,filed Apr. 28, 2004.

BACKGROUND OF THE INVENTION

Adenovirus is a double-stranded DNA virus with a genome size of about 36kilobases (kb), which has been widely used for gene transferapplications due to its ability to achieve highly efficient genetransfer in a variety of target tissues and large transgene capacity.Conventionally, E1 genes of adenovirus are deleted and replaced with atransgene cassette consisting of the promoter of choice, cDNA sequenceof the gene of interest and a poly A signal, resulting in a replicationdefective recombinant virus.

Adenoviruses have a characteristic morphology with an icosahedral capsidconsisting of three major proteins, hexon (II), penton base (III) and aknobbed fibre (IV), along with a number of other minor proteins, VI,VIII, IX, IIIa and IVa2 [W. C. Russell, J. Gen Virol., 81:2573-2604(November 2000)]. The virus genome is a linear, double-stranded DNAhaving inverted terminal repeats (ITRs), with a terminal proteinattached covalently to the 5′ termini. The virus DNA is intimatelyassociated with the highly basic protein VII and a small peptide termedmu. Another protein, V, is packaged with this DNA-protein complex andprovides a structural link to the capsid via protein VI. The virus alsocontains a virus-encoded protease, which is necessary for processing ofsome of the structural proteins to produce mature infectious virus.

Recombinant adenoviruses have been described for delivery of moleculesto host cells to induce an immune response. See, U.S. Pat. No.6,083,716, which provides adenoviral vectors derived from the twochimpanzee adenoviruses, C1 and C68 (also termed Pan 9) andInternational Patent Publication No. WO 02/33645 [Pan 5, Pan6,Pan7-derived vectors].

What is needed in the vaccine field is method of immunizing that willinduce a strong immune response with minimal responses to the vaccinecarrier.

SUMMARY OF THE INVENTION

The methods of the invention involve sequentially delivering one or moreselected heterologous gene(s) to a mammalian patient via an adenoviralvector and an adeno-associated virus vector. Each of the vectorsexpresses the same immunogenic or antigenic product or a cross-reactiveproduct.

The method of the invention provides a boosting of the immune responseto the product carried by the viral vectors.

These and other embodiments and advantages of the invention aredescribed in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the total IgG responses to EboZ GP after heterologousprime and boost. B10Br mice were IM immunized with 5×10¹⁰, 5×10⁹, or5×10⁸ particles/mouse of H5GP vector. Six weeks later, mice were boostedIM with 5×10¹⁰ genome copies/mouse of AAV2/1 GP vector. Serum from 3mice in each group were collected and pooled at either day 42 afterinjection (pre-boost), or 1 week after boost, or 2 weeks after boost, asindicated in the FIGURE. Total IgG responses to GP were measured byELISA.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of specifically inducing an immuneresponse by administering an adeno-associated virus (AAV) vector and anadenoviral (Ad) vector. Each of the viral vectors contains aheterologous expression cassette comprising a nucleic acid sequenceencoding a product for inducing an immune response under the control ofregulatory control sequences which direct expression of the product. Inone embodiment, the encoded products are the same in order to provide aprime/boost effect to the product, which induces immunity to thepathogen from which the product is derived or a cross-reactive pathogen.However, the regulatory control elements and other viral elements candiffer in the adenoviruses administered. In another embodiment, theencoded products differ, but provide a cross-reactive effect.

Unexpectedly, the inventors have found that a significantantigen-specific humoral response is observed in a regimen involvingsequential rAd-mediated and rAAV-mediated administration of the antigen.Without wishing to be bound by theory, it is believed that an enhanced Tcell response may also be observed, depending upon the antigen and routeof administration.

Thus, the method of the invention involves sequential administration ofan adenoviral vector carrying a heterologous expression cassette and anAAV vector carrying a heterologous expression cassette, as describedherein. As used herein, sequential administration encompasses deliveryof an adenoviral vector followed by delivery of an AAV vector, i.e., ata time period apart from one another. Sequential administration furtherencompasses delivery of an AAV vector followed by delivery of anadenoviral vector. The method of the invention also encompassescoadministration of an adenoviral vector and an AAV vector, i.e.,administration simultaneously or in close time proximity to each other,whether formulated together or separately. In one embodiment, sequentialdelivery of the antigenic product can be achieved even uponco-administration of different vectors due to the kinetics of antigenexpression via one of the vectors, the use of an inducible promoter, orsome other activatable expression system.

In one embodiment, the immunization regimen of the invention providesadministration of multiple adenoviral vectors in combination withsequential administration of an AAV vector as described herein. Inanother embodiment, the regimen of the invention provides administrationof an adenoviral vector in combination with sequential administration ofmultiple AAV vectors. In yet another embodiment, the regimen providessequential administration of multiple adenoviral vectors in combinationwith multiple AAV vectors.

In the present invention, a regimen that involves administration of morethan one adenoviral vector typically utilizes a first adenoviral vectorhaving a capsid protein of a first serotype and administration of atleast one additional adenoviral vector that has a capsid protein whichis immunologically distinct from the prior adenoviral vector(s).

As used herein, a capsid protein is immunologically distinct fromanother capsid protein if it can be administered to a subject in theabsence of an immune response which prevents infection with a secondcapsid protein (e.g., a clearing neutralizing antibody response).Suitably, the capsid proteins of a boosting vector(s) are from aserologically distinct source from the capsid protein of previouslyadministered (priming) vector(s). However, in other embodiments, thecapsid proteins of the priming (and optionally, boosting) vectors can bedelivered without regard to serological distinctiveness, if the nativeantibody epitopes of the capsid proteins are masked, modified, orotherwise neutralized (e.g., by co-administration of an exogenousmolecule).

Similarly, a regimen that involves administration of more than one AAVvector typically utilizes a first AAV vector having a capsid protein ofa first serotype and administration of at least one additional AAVvector that has a capsid protein that is immunologically distinct fromthe prior AAV vector(s).

Depending upon the desired routes of administration, one of skill in theart can select an appropriate regimen. For example, peak immune responseis generally observed about 10 to 14 days following an Ad-mediateddelivery. However, boosting following this peak may generate a secondpeak. Thus, it may be desirable to time expression of a boosting antigento express from about 10 to 21 days, or 18 to 28 days, or 28 days to 6months following Ad-mediated delivery. In another example, certain AAVserotypes demonstrate peak expression about 3 to 4 weeks followingdelivery. Thus, boosting following AAV delivery may be timed to expressthe antigen, or a cross-reactive antigen, about 3 weeks to about 4weeks, about 4 weeks to about 2 months, or about 2 months to 6 months,or longer, following AAV delivery.

In one embodiment, the heterologous expression cassette delivered via aviral vector, e.g., an AAV vector, according to the invention containsthe immunogenic product operably linked to an inducible or regulatablepromoter. When used in a regimen according to the present invention, theinducing or regulating agent is typically administered such thatexpression of product is activated immediately upon administration ofthe viral vector. Thereafter, expression can be extinguished by removalof the inducing or regulating agent. The regimen of the invention caninclude “pulse” activation of expression. Thus, the method of theinvention permits expression to be induced, extinguished, and then againinduced after a period of time. In another embodiment, expression isinduced not upon administration, but several days to several weeksfollowing administration. This embodiment may permit co-delivery of anAd and AAV followed by induction (activation) several weeks followingdelivery, depending upon the delay caused by the induction agent. Forexample, once an inducing agent is delivered, it may be 7 to 10 days, orlonger before the effect is observed. One of skill in the art will befamiliar with the delay between delivery of the inducing or activatingagent and the effect and will be able to readily factor this into theselected regimen.

Optionally, a regimen of the invention can be combined withadministration of other immunogenic or therapeutic moieties including,e.g., DNA molecules encoding a desired immunogenic or therapeuticmolecule, or a protein-based immunogen or therapeutic.

These and other aspect of the regimen of the invention, and the productsof the invention, are described in more detail.

I. Adenoviral Vectors

The term “functionally deleted” or “functional deletion” means that asufficient amount of the gene region is removed or otherwise damaged,e.g., by mutation or modification, so that the gene region is no longercapable of producing functional products of gene expression. If desired,the entire gene region may be removed. Other suitable sites for genedisruption or deletion are discussed elsewhere in the application.

A. Serotypes

Suitably, the adenoviral vectors of the invention contain one or moreadenoviral elements derived from a selected adenoviral genome. In oneembodiment, the vectors contain adenoviral inverted terminal repeat(ITRs) from one selected serotype and additional adenoviral sequencesfrom the same adenoviral serotype. In another embodiment, the vectorscontain adenoviral sequences that are derived from a differentadenoviral serotype than that which provides the ITRs. As definedherein, a pseudotyped adenovirus refers to an adenovirus in which thecapsid protein of the adenovirus is from a different serotype than theserotype which provides the ITRs.

The selection of the serotype of the ITRs, any adenoviral genes present,the capsid protein, and the serotype of any other adenoviral sequencespresent in vector is not a limitation of the present invention. Avariety of adenovirus strains are available from the American TypeCulture Collection, Manassas, Va., or available by request from avariety of commercial and institutional sources. Further, the sequencesof many such strains are available from a variety of databasesincluding, e.g., PubMed and GenBank. Adenovirus vectors prepared fromother simian or from human adenoviruses are described in the publishedliterature [see, for example, U.S. Pat. No. 5,240,846]. The DNAsequences of a number of adenovirus types are available from theGenBank™ database, including type Ad5 [GenBank™ Accession No. M73260].The adenovirus sequences may be obtained from any known adenovirusserotype, such as serotypes C, D, 1-40, and particularly 2, 3, 4, 5, 7,12 and 40, and further including any of the presently identified humantypes. Similarly adenoviruses known to infect non-human animals (e.g.,simians) may also be employed in the vector constructs of thisinvention. See, e.g., U.S. Pat. No. 6,083,716. Examples of otheradenovirus serotype that may be useful in the method of the inventionincludes, e.g., serotype 34 [WO 2004/4097016], serotype 24 [WO2004/083418]; and serotype 35 [EP 1054064].

The viral sequences, helper viruses, if needed, and recombinant viralparticles, and other vector components and sequences employed in theconstruction of the vectors described herein are obtained as describedabove. The DNA sequences of the simian adenovirus sequences of theinvention are employed to construct vectors and cell lines useful in thepreparation of such vectors.

In one embodiment, at least one of the adenoviruses used in theinvention contains a capsid derived from a non-human primate. Examplesof suitable non-human primate sequences include simian adenoviruses,such as, Pan5 (also C5), Pan6 (also C6), Pan7 (also C7), SV1, SV25, SV39[see, WO 02/33645, incorporated by reference], and Pan 9 (also C68) andC1 [U.S. Pat. No. 6,083,716, incorporated by reference], and SA 18 [U.S.patent application Ser. No. 10/465,302 and its international counterpartWO 2005/001103, incorporated by reference].

The invention further encompasses pseudotyped adenoviruses, chimeric andhybrid adenoviral vectors. See, e.g., U.S. patent application Ser. No.10/465,302 and its international counterpart WO 2005/001103,incorporated by reference.

However, the invention is not limited to the selection of the capsidserotype or the origin of the other adenoviral elements present in theviral vector.

B. Adenoviral Elements

The adenoviral particles or vectors used in the present invention arecomposed of adenovirus capsids having packaged therein an expressioncassette carrying a product to be expressed in the host and sufficientviral elements to permit delivery of the expression cassette to aninfected host cell. These adenoviral vectors can bereplication-defective, thereby avoiding replication in a host cell.

In one embodiment, adenoviral particles used in the invention contain 5′adenoviral cis-elements and 3′ adenoviral cis-elements at the extreme 5′and 3′ termini of the adenovirus, respectively. The 5′ end of theadenoviral genome contains the 5′ cis-elements necessary for packagingand replication; i.e., the 5′ inverted terminal repeat (ITR) sequences(which functions as origins of replication) and the 5′ packagingenhancer domains (that contain sequences necessary for packaging linearAd genomes and enhancer elements for the E1 promoter). The 3′ end of theadenoviral genome includes the 3′ cis-elements (including the ITRs)necessary for packaging and encapsidation.

The adenoviral vectors can contain functional adenoviral genes native toan adenoviral genome, or can contain multiple deletions relative to anative adenoviral genome. In one embodiment, the adenovirus has afunctional deletion in the E1 region that renders itreplication-defective and incapable of expressing the gene products ofthis region, including the E1a and E1b gene products.

In addition, the adenoviral vectors used in the invention can befunctionally deleted in one or more of the other adenoviral early generegions. For example, an adenoviral vector used in the invention cancontain a functional deletion in the E1 region and, optionally, afunctional deletion in one or more of the adenovirus E2 region, theadenovirus E3 region, or the adenovirus E4 gene region. Functionaldeletions in the E2 region may include destruction of the ability toexpress functional one or both of the E2 product, E2a and the E2b.Similarly, a functional deletion in the E4 region may includedestruction of the ability to express functional ORF1, ORF2, ORF3, ORF4,ORF5, ORF6, ORF7 gene products.

Elimination of E3 permits insertion of an expression cassette in thatregion. However, E3 is believed to be implicated in modulation of hostimmune response to the adenovirus, and thus, in another embodiment, isretained. In this embodiment, the E3 gene product is expressed under thecontrol of a heterologous promoter, to avoid down-regulation of thenative E3 promoter which requires E1 expression.

The adenoviruses used in the invention that contain an E4-deletion arefunctionally deleted of one or more of the open reading frames (ORFs) ofE4 (e.g., ORF 1, ORF2, ORF3, ORF4, ORF5, ORF6 and ORF7). In oneembodiment, the construct contains a functional deletion of each of theE4 ORFs. In another embodiment, all E4 ORFs are deleted with theexception of E4 ORF6. In still another embodiment, another combinationof one or more of these E4 ORFs is functionally deleted in theadenoviral construct used in the method of the invention.

An adenoviral vector used in the invention can be functionally deletedin one or more other adenoviral gene regions, e.g., in one or more ofthe adenoviral intermediate genes, genes IX and IVa₂, or adenoviral lategenes (L1, L2, L3, L4 and L5).

In still other embodiments, other deletions may be made in the otherstructural or non-structural adenovirus genes. These deletions can beused individually, i.e., an adenovirus sequence for use in the presentinvention may contain deletions in only a single region.

Methods for generating adenoviral vectors containing functionaldeletions that eliminate the ability of the vector to express proteinsrequired for packaging into an adenoviral capsid are known to those ofskill in the art and are not a limitation of the present invention.

C. Vector Elements

The methods employed for the selection of the antigenic or immunogenictarget (i.e., product) and the sequences encoding same, the cloning andconstruction of the “heterologous expression cassette” and its insertioninto the viral vector are within the skill in the art given theteachings provided herein. According to the present invention, theheterologous expression cassette can be located in the site of anyadenoviral region relative to a native adenoviral genome, which islocated between the 5′ and 3′ adenovirus ITRs. In one embodiment, theheterologous expression cassette is located in the native E1 region ofthe adenoviral vector. In another embodiment, the heterologousexpression cassette is located in the native E3 region. In otherembodiments, the gene product is expressed from the native E2 region ofthe adenoviral vector or from the native E4 region of the adenoviralvector, and is operably linked to regulatory control elements which arenon-contiguous with the sequences encoding the gene product. Theinvention is not limited to the orientation of the heterologousexpression cassette, which may be inserted 5′-3′ or 3′-5′, relative theorientation of the adenoviral genome flanking the expression cassette.

In yet another embodiment, the adenoviral vector carries more than oneheterologous expression cassette, which can be inserted into multipledeletion sites in the adenoviral genome. In this embodiment, theheterologous expression cassette can express the same or differentproducts from multiple locations in the adenoviral genome.

1. The Nucleic Acid Sequence

The expression cassette contains a nucleic acid sequence, heterologousto the vector sequences flanking the sequence, which encodes apolypeptide, protein, or other product, of interest. Suitably, thisproduct is immunogenic or antigenic. The nucleic acid coding sequence isoperatively linked to regulatory components in a manner which permitstranscription, translation, and/or expression of the product in a hostcell. Suitable nucleic acid sequences and products may be readilyselected by one of skill in the art. The selection of these elements isnot a limitation of this invention.

2. Regulatory Elements

In addition to the major elements identified above for the expressioncassette, the vector also includes conventional control elements whichare operably linked to the sequences encoding the product in a mannerthat permits transcription, translation and/or expression of the productin a cell infected with the virus used in the invention. As used herein,“operably linked” sequences include both expression control sequencesthat are contiguous with the product (e.g., gene) of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product. A great number of expression controlsequences, including promoters which are native, constitutive, inducibleand/or tissue-specific, are known in the art and may be utilized.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer and/or CMV intronic region) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter an its variants, the phosphoglycerolkinase (PGK) promoter, and the EF1α promoter [Invitrogen]. In oneembodiment, in which the antigen is a severe acute respiratory syndrome(SARS) coronavirus antigen, the β-actin promoter is used in combinationwith an enhancer and intron regions from CMV.

In another embodiment, the native promoter for the gene will be used.The native promoter may be preferred when it is desired that expressionof the product should mimic native expression. The native promoter maybe used when expression of the product must be regulated temporally ordevelopmentally, in a tissue-specific manner, or in response to specifictranscriptional stimuli. In a further embodiment, other nativeexpression control elements, such as enhancer elements, polyadenylationsites or Kozak consensus sequences may also be used to mimic the nativeexpression.

Another embodiment of the expression cassette includes a nucleic acidsequence encoding a product operably linked to a tissue-specificpromoter. For instance, if expression in skeletal muscle is desired, apromoter active in muscle should be used. These include the promotersfrom genes encoding skeletal β-actin, myosin light chain 2A, dystrophin,muscle creatine kinase, as well as synthetic muscle promoters withactivities higher than naturally occurring promoters (see Li et al.,Nat. Biotech., 17:241-245 (1999)). Other examples of promoters that aretissue-specific are known for liver [albumin, Miyatake et al., J.Virol., 71:5124-32 (1997); hepatitis B virus core promoter, Sandig etal., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein (AFP), Arbuthnot etal., Hum. Gene Ther., 7:1503-14 (1996)], bone osteocalcin [Stein et al.,Mol. Biol. Rep., 24:185-96 (1997)]; bone sialoprotein [Chen et al., J.Bone Miner. Res., 11:654-64 (1996)], lymphocytes [CD2, Hansal et al., J.Immunol., 161:1063-8 (1998); immunoglobulin heavy chain; T cell receptorchain], neuronal such as neuron-specific enolase (NSE) promoter(Andersen et al., Cell. Mol. Neurobiol., 13:503-15 (1993)),neurofilament light-chain gene (Piccioli et al., Proc. Natl. Acad. Sci.USA, 88:5611-5 (1991)), and the neuron-specific vgfgene (Piccioli etal., Neuron, 15:373-84 (1995)), among others.

Optionally, vectors carrying sequences encoding immunogenic products mayalso include selectable markers or reporter genes may include sequencesencoding geneticin, hygromycin or puromycin resistance, among others.Such selectable reporters or marker genes (preferably located outsidethe viral genome to be packaged into a viral particle) can be used tosignal the presence of the plasmids in bacterial cells, such asampicillin resistance. Other components of the vector may include anorigin of replication. Selection of these and other promoters and vectorelements is conventional for those of skill in the art and many suchsequences are available [see, e.g., Sambrook et al, and references citedtherein].

These vectors are generated using the techniques and sequences providedherein, in conjunction with techniques known to those of skill in theart. Such techniques include conventional cloning techniques of cDNAsuch as those described in texts [Sambrook et al, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.],use of overlapping oligonucleotide sequences of the adenovirus genomes,polymerase chain reaction, and any suitable method which provides thedesired nucleotide sequence.

As stated above, while in one embodiment the immunization regimen of theinvention involves sequential delivery of the same immunogenic productvia different viral vectors, the heterologous expression cassette(s)used in the vectors of any given regimen need not be the same. In fact,each heterologous expression cassette can contain regulatory sequencesfor the immunogenic product and/or vector elements which differ from theregulatory sequences and vector element in other expression cassettesused in the regimen. Thus, the selection of these regulatory and vectorelements is not a limitation of the invention, even within the contextof an immunization regimen for a selected subject.

II. Adenoviral Particles

A variety of production methods for adenoviral particles is known tothose of skill in the art. The selection of appropriate productionmethods is not a limitation of the present invention. See, e.g., U.S.Pat. No. 6,083,716; International Patent Publication No. WO 02/33645;and U.S. patent application Ser. No. 10/465,302, which are incorporatedby reference. Briefly, production of an adenoviral vector lacking theability to express one or more essential adenoviral products (e.g., E1a,E1b, E2a, E2b, E4 ORF6, L1, L2, L3, L4 and L5) can be cultured in thepresence of the missing adenoviral gene products which are required forviral infectivity and propagation of an adenoviral particle. Thesehelper functions may be provided by culturing the adenoviral vector inthe presence of one or more helper constructs (e.g., a plasmid or virus)and/or a packaging host cell. See, for example, the techniques describedfor preparation of a “minimal” human Ad vector in International PatentPublication No. WO96/13597, published May 9, 1996, and incorporatedherein by reference.

Regardless of whether the adenoviral vectors contains only the minimalAd sequences, or the entire Ad genome with only functional deletions inthe E1, E3, and/or E4 regions, in one embodiment, the recombinant viruscontains a capsid derived from a single adenovirus. Alternatively, inother embodiments, recombinant pseudotyped adenoviruses or hybridadenoviruses may be used in the methods of the invention. Methods forproducing these adenoviral vectors have been described.

A. Helper Viruses

Thus, depending upon the adenovirus gene content of the viral vectorsemployed to carry the expression cassette, a helper adenovirus ornon-replicating virus fragment may be necessary to provide sufficientadenovirus gene sequences necessary to produce an infective recombinantviral particle containing the expression cassette. Useful helper virusescontain selected adenovirus gene sequences not present in the adenovirusvector construct and/or not expressed by the packaging cell line inwhich the vector is transfected. In one embodiment, the helper virus isreplication-defective and contains a variety of adenovirus genes inaddition to the sequences described above. Such a helper virus isdesirably used in combination with an E1-expressing cell line.

Helper viruses may also be formed into poly-cation conjugates asdescribed in Wu et al, J. Biol. Chem., 264:16985-16987 (1989); K. J.Fisher and J. M. Wilson, Biochem. J, 299:49 (Apr. 1, 1994). Helper virusmay optionally contain a second reporter expression cassette. A numberof such reporter genes are known to the art. The presence of a reportergene on the helper virus which is different from the gene product on theadenovirus vector allows both the Ad vector and the helper virus to beindependently monitored. This second reporter is used to enableseparation between the resulting recombinant virus and the helper virusupon purification.

B. Complementation Cell Lines

To generate recombinant adenoviruses (Ad) deleted in any of the genesdescribed above, the function of the deleted gene region, if essentialto the replication and infectivity of the virus, must be supplied to therecombinant virus by a helper virus or cell line, i.e., acomplementation or packaging cell line. In many circumstances, a cellline expressing the human adenovirus E1 can be used to transcomplementthe chimp Ad vector. This is particularly advantageous because, due tothe diversity between the chimp Ad sequences of the invention and thehuman AdE1 sequences found in currently available packaging cells, theuse of the current human E1-containing cells prevents the generation ofreplication-competent adenoviruses during the replication and productionprocess. However, in certain circumstances, it will be desirable toutilize a cell line that expresses the E1 gene products can be utilizedfor production of an E1-deleted simian adenovirus. Such cell lines havebeen described. See, e.g., U.S. Pat. No. 6,083,716.

One may utilize the sequences provided herein to generate a packagingcell or cell line that expresses, at a minimum, the adenovirus E1 geneunder the transcriptional control of a promoter for expression in aselected parent cell line. Inducible or constitutive promoters may beemployed for this purpose. Examples of such promoters are described indetail elsewhere in this specification. A parent cell is selected forthe generation of a novel cell line expressing any desired Ad gene.Without limitation, such a parent cell line may be HeLa [ATCC AccessionNo. CCL 2], A549 [ATCC Accession No. CCL 185], HEK 293, KB [CCL 17],Detroit [e.g., Detroit 510, CCL 72] and WI-38 [CCL 75] cells, amongothers. These cell lines are all available from the American TypeCulture Collection, 10801 University Boulevard, Manassas, Va.20110-2209. Other suitable parent cell lines may be obtained from othersources.

Such E1-expressing cell lines are useful in the generation ofrecombinant adenovirus E1 deleted vectors. Additionally, oralternatively, the invention provides cell lines expressing one or moresimian adenoviral gene products, e.g., E1a, E1b, E2a, and/or E4 ORF6,that can be constructed using essentially the same procedures for use inthe generation of recombinant simian viral vectors. Such cell lines canbe utilized to transcomplement adenovirus vectors deleted in theessential genes that encode those products. The preparation of a hostcell according to this invention involves techniques such as assembly ofselected DNA sequences. This assembly may be accomplished utilizingdirect cloning techniques. Such techniques have been described [G. Gaoet al, Gene Ther. 2003 October; 10(22):1926-1930; US Patent PublicationNo. 2003-0092161-A, published May 15, 2003; International PatentApplication No. PCT/US03/12405]. Alternatively, techniques such as cDNAand genomic cloning, which are well known and are described in Sambrooket al., cited above, and use of overlapping oligonucleotide sequences ofthe adenovirus genomes, combined with polymerase chain reaction,synthetic methods, and any other suitable methods which provide thedesired nucleotide sequence can be used.

In still another alternative, the essential adenoviral gene products areprovided in trans by the adenoviral vector and/or helper virus. In suchan instance, a suitable host cell can be selected from any biologicalorganism, including prokaryotic (e.g., bacterial) cells, and eukaryoticcells, including, insect cells, yeast cells and mammalian cells. Hostcells are selected from among any mammalian species, including, withoutlimitation, cells such as A549, WEHI, 3T3, 10T1/2, HEK 293 cells orPERC6 (both of which express functional adenoviral E1) [Fallaux, F J etal, (1998), Hum Gene Ther, 9:1909-1917], Saos, C2C12, L cells, HT1080,HepG2 and primary fibroblast, hepatocyte and myoblast cells derived frommammals including human, monkey, mouse, rat, rabbit, and hamster. Theselection of the mammalian species providing the cells is not alimitation of this invention; nor is the type of mammalian cell, i.e.,fibroblast, hepatocyte, tumor cell, etc.

C. Assembly of Viral Particle and Transfection of a Cell Line

Generally, when delivering the vector comprising the expression cassetteby transfection, the vector is delivered in an amount from about 5 μg toabout 100 μg DNA, or about 10 to about 50 μg DNA to about 1×10⁴ cells toabout 1×10¹³ cells, or about 10⁵ cells. However, the relative amounts ofvector DNA to host cells may be adjusted, taking into consideration suchfactors as the selected vector, the delivery method and the host cellsselected.

The vector may be any vector known in the art or disclosed above,including naked DNA, a plasmid, phage, transposon, cosmids, episomes,viruses, etc. Introduction into the host cell of the vector may beachieved by any means known in the art or as disclosed above, includingtransfection, and infection. One or more of the adenoviral genes may bestably integrated into the genome of the host cell, stably expressed asepisomes, or expressed transiently. The gene products may all beexpressed transiently, on an episome or stably integrated, or some ofthe gene products may be expressed stably while others are expressedtransiently.

Furthermore, the promoters for each of the adenoviral genes may beselected independently from a constitutive promoter, an induciblepromoter or a native adenoviral promoter. The promoters may be regulatedby, for example, a specific physiological state of the organism or cell(i.e., by the differentiation state or in replicating or quiescentcells) or by exogenously-added factors, for example. Introduction of themolecules (e.g., plasmids or viruses) into the host cell may also beaccomplished using techniques known to the skilled artisan and asdiscussed throughout the specification. In preferred embodiment,standard transfection techniques are used, e.g., CaPO₄ transfection orelectroporation.

Assembly of the selected DNA sequences of the adenovirus (as well as thetransgene and other vector elements into various intermediate plasmids,and the use of the plasmids and vectors to produce a recombinant viralparticle are all achieved using conventional techniques. Such techniquesinclude conventional cloning techniques of cDNA such as those describedin texts [Sambrook et al, cited above], use of overlappingoligonucleotide sequences of the adenovirus genomes, polymerase chainreaction, and any suitable method which provides the desired nucleotidesequence. Standard transfection and co-transfection techniques areemployed, e.g., CaPO₄ precipitation techniques. Other conventionalmethods employed include homologous recombination of the viral genomes,plaquing of viruses in agar overlay, methods of measuring signalgeneration, and the like.

For example, following the construction and assembly of the desiredexpression cassette-containing viral vector, the vector is transfectedin vitro in the presence of a helper virus into the packaging cell line.Homologous recombination occurs between the helper and the vectorsequences, which permits the adenovirus-transgene sequences in thevector to be replicated and packaged into virion capsids, resulting inrecombinant viral vector particles. The current method for producingsuch virus particles is transfection-based. However, the invention isnot limited to such methods.

The resulting recombinant viruses are useful in transferring a selectedtransgene to a selected cell.

III. MV Vectors

In one embodiment, the AAV vectors useful in the invention contain, at aminimum, sequences encoding an AAV capsid in which is packaged aheterologous expression cassette. The cassette is flanked by AAV 5′ ITRand AAV 3′ ITR. The AAV ITRs may be from the same source as the capsid,or from a different source (i.e., a pseudotyped AAV vector).

In one aspect, the invention provides a method of generating arecombinant adeno-associated virus (AAV). Such a method involvesculturing a host cell which contains a nucleic acid sequence encoding anadeno-associated virus (AAV) capsid protein, or fragment thereof, asdefined herein; a functional rep gene; an expression cassette flankedby, at a minimum, AAV inverted terminal repeats (ITRs); and sufficienthelper functions to permit packaging of the expression cassette into theAAV capsid.

The components required to be cultured in the host cell to package anexpression cassette flanked by AAV ITRs in an AAV capsid may be providedto the host cell in trans. Alternatively, any one or more of therequired components (e.g., expression cassette, rep sequences, capsequences, and/or helper functions) may be provided by a stable hostcell which has been engineered to contain one or more of the requiredcomponents using methods known to those of skill in the art. Mostsuitably, such a stable host cell will contain the required component(s)under the control of an inducible promoter. However, the requiredcomponent(s) may be under the control of a constitutive promoter.Examples of suitable inducible and constitutive promoters are providedherein, in the discussion of regulatory elements suitable for use withthe sequences encoding the product. In still another alternative, aselected stable host cell may contain selected component(s) under thecontrol of a constitutive promoter and other selected component(s) underthe control of one or more inducible promoters. For example, a stablehost cell may be generated that is derived from 293 cells (which containE1 helper functions under the control of a constitutive promoter), butwhich contains the rep and/or cap proteins under the control ofinducible promoters. Still other stable host cells may be generated byone of skill in the art.

The expression cassette, rep sequences, cap sequences, and helperfunctions required for producing the rAAV of the invention may bedelivered to the packaging host cell in the form of any genetic elementwhich transfer the sequences carried thereon. The selected geneticelement may be delivered by any suitable method, including thosedescribed herein. The methods used to construct any embodiment of thisinvention are known to those with skill in nucleic acid manipulation andinclude genetic engineering, recombinant engineering, and synthetictechniques. See, e.g., Sambrook et al, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. Similarly,methods of generating rAAV virions are well known and the selection of asuitable method is not a limitation on the present invention. See, e.g.,K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No.5,478,745.

Unless otherwise specified, the AAV ITRs, AAV capsids, and otherselected AAV components described herein, may be readily selected fromamong any AAV serotype, including, without limitation, AAV1[International Patent Publication No. WO 00/28061], AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9 and one of the other AAV described inInternational Patent Publication No. WO 2003/042397, U.S. PatentApplication No. 60/508,226 and its international counterpartPCT/US04/028817, and U.S. Patent Application No. 60/669,083, entitled“Method of Increasing the Function of an AAV Vector”, filed Apr. 7,2005, which are incorporated by reference herein. These ITRs or otherAAV components may be readily isolated using techniques available tothose of skill in the art from an AAV serotype. Such AAV may be isolatedor obtained from academic, commercial, or public sources (e.g., theAmerican Type Culture Collection, Manassas, Va.). Alternatively, the AAVsequences may be obtained through synthetic or other suitable means byreference to published sequences such as are available in the literatureor in databases such as, e.g., the GenBank™ database, PubMed™ database,or the like.

The AAV vectors useful in the invention include vectors in which all AAVsequences and capsid proteins (e.g., fiber, penton, hexon) are derivedfrom a single source. Alternatively, AAV vectors used in the inventioninclude those that are trans-encapsidated (i.e., contain an AAV capsidof a different source than the AAV ITRS and/or other AAV elements in thecapsid). Such trans-encapsidated vectors include those that arepseudotyped using conventional techniques, or that contain synthetic,artificial and/or chimeric capsids. See, e.g., International PatentPublication No. WO 2003/042397; U.S. Patent Application No. 60/508,226and its international counterpart PCT/US04/028817, and U.S. Pat. No.6,491,907].

In one embodiment, the invention utilized AAV viral vectors in which oneor more of the vp1, vp2 and/or vp3 proteins (which assemble to form anAAV capsid) is modified in order to enhance antigenicity of the AAV,e.g., by increasing vector uptake by and activation of antigenpresenting cells (APCs), dendritic cells, or another target cell ortissue type. The modified vp1, vp2 or vp3 protein assembles into an AAVcapsid having the inserted peptide or protein exposed on the outersurface of the capsid. These modifications may be made using the methodsof Girod et al, [A. Girod, et. al., Nat Med, 5(9):1052-1056 (1999)] orWarrington et al, [K H. Warrington, Jr., et. al., J. Virol.,78(12):6595-6609 (2004)]. According to the methods of Girod, a site inthe vp3 coding domain of a selected AAV can by modified by insertingnucleic acid sequences at the coding region corresponding to amino acidposition 587 of AAV2 [and in the corresponding sequence of other AAVs,as determined upon alignment using conventional programs and methods].Such an amino acid insert may be from about 1 to 34 amino acids inlength and may provide the assembled AAV capsid containing the modifiedAAV vp2 protein with a desired peptide, e.g., a peptide specificreceptor, antigen, or another desirable sequence. Examples of suitablefragments include, e.g., peptides targeted to dendritic cells, such asDC3, DC12, and DC18 those described in T. J. Curiel, et al., J. Immunol,172 (120):7425-31 (2004).

According to the methods of Warrington, the N-terminus of the vp2 capsid(i.e., position 138 of AAV2 and corresponding positions in other AAVsequences) can be modified to contain an insertion of a peptide,resulting in a fusion protein useful in the assembled AAV capsidcontaining the modified capsid protein. In one embodiment, these fusionproteins confer additional targeting sequences. In other embodiments,the fusion proteins may provide additional antigenic sequences. In oneembodiment, insertions of up to 250 amino acids, more desirably 150amino acids, or of lengths from 5 to 150 amino acids are selected.Examples of suitable sources of peptides include fragments obtainedfrom, e.g., flagellin, heat shock protein, and complement proteins (suchas complement component 3, fragment d).

It will be understood that still other AAV vectors may be selected foruse in the present invention without limitation as to the origin of thecapsid and/or the source of the AAV ITRs or other AAV elements.

A. The Packaging Expression Cassette

A heterologous expression cassette as defined above in connection withthe adenoviral vectors useful in the invention is located between, atits 5′ end, 5′ AAV inverted terminal repeats (ITRs) and, at its 3′terminus, 3′ AAV ITRs. Other AAV or vector elements may also be locatedbetween the expression cassette and ether the 5′ AAV ITRs or the 3′ AAVITRs. It is this construct that is packaged into a capsid protein toprovide an AAV vector of the invention.

The elements of the heterologous expression cassette, including thenucleic acid sequences encoding the product, the vector elements, andthe like, are readily selected from among those described above of theadenoviral vector.

In one embodiment, the heterologous expression cassette delivered viarAAV according to the invention contains the immunogenic productoperably linked to an inducible or regulatable promoter. When used in aregimen according to the present invention, the inducing or regulatingagent is typically administered such that expression of product isactivated immediately following administration of the rAAV. Thereafter,expression can be extinguished by removal of the inducing or regulatingagent. The regimen of the invention can include “pulse” activation ofexpression. In other words, expression can be induced, extinguished, andthen again induced after a period of time.

Inducible and regulatable promoters allow regulation of gene expressionand can be regulated by exogenously supplied compounds, environmentalfactors such as temperature, or the presence of a specific physiologicalstate, e.g., acute phase, a particular differentiation state of thecell, or replication. Regulatable promoters and systems are availablefrom a variety of commercial sources, including, without limitation,Invitrogen, Clontech and Ariad. Many other systems have been describedand can be readily selected by one of skill in the art. For example,inducible promoters include the zinc-inducible sheep metallothionine(MT) promoter and the dexamethasone (Dex)-inducible mouse mammary tumorvirus (MMTV) promoter. Other inducible systems include the T7 polymerasepromoter system [WO 98/10088]; the ecdysone insect promoter [No et al,Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)], thetetracycline-repressible system [Gossen et al, Proc. Natl. Acad. Sci.USA, 89:5547-5551 (1992)], and the tetracycline-inducible system [Gossenet al, Science, 268-1766-1769 (1995), see also Harvey et al, Curr. Opin.Chem. Biol., 2:512-518 (1998)]. Other systems include the FK506 dimer,VP16 or p65 using castradiol, diphenol murislerone, the RU486-induciblesystem [Wang et al, Nat. Biotech., 15:239-243 (1997) and Wang et al,Gene Ther., 4:432-441 (1997)] and the rapamycin-inducible system [Magariet al, J. Clin. Invest., 100:2865-2872 (1997)]. The effectiveness ofsome inducible promoters increases over time. In such cases one canenhance the effectiveness of such systems by inserting multiplerepressors in tandem, e.g., TetR linked to a TetR by an IRES.Alternatively, one can wait at least 3 days before screening for thedesired function. One can enhance expression of desired proteins byknown means to enhance the effectiveness of this system. For example,using the Woodchuck Hepatitis Virus Post-transcriptional RegulatoryElement (WPRE). Selection of the regulatable promoter is not alimitation of the present invention.

Further, the method of the invention is not limited to use of suchpromoters, and the heterologous expression cassettes carried by the rAAVaccording to the method of the invention can include constitutive,native, or tissue-specific promoter. Selection of this promoter is not alimitation on the present invention.

B. Rep and Cap Sequences

In addition to the minigene, the host cell contains the sequences whichdrive expression of an AAV capsid protein of the invention (or a capsidprotein comprising a fragment thereof) in the host cell and repsequences of the same serotype as the serotype of the AAV ITRs found inthe minigene, or a cross-complementing serotype. The AAV cap and repsequences may be independently obtained from an AAV source as describedabove and may be introduced into the host cell in any manner known toone in the art as described above. Additionally, when pseudotyping anAAV vector in (e.g., an AAV9/HU.14 capsid), the sequences encoding eachof the essential rep proteins may be supplied by different AAV serotypes(e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or any ofthe serotypes identified herein). For example, the rep78/68 sequencesmay be from AAV2, while the rep52/40 sequences may be from AAV8.

In one embodiment, the host cell stably contains the capsid proteinunder the control of a suitable promoter, such as those described above.In one embodiment, the capsid protein is expressed under the control ofan inducible promoter. In another embodiment, the capsid protein issupplied to the host cell in trans. When delivered to the host cell intrans, the capsid protein may be delivered via a plasmid which containsthe sequences necessary to direct expression of the selected capsidprotein in the host cell. When delivered to the host cell in trans, theplasmid carrying the capsid protein may also carry other sequencesrequired for packaging the rAAV, e.g., the rep sequences.

In another embodiment, the host cell stably contains the rep sequencesunder the control of a suitable promoter, such as those described above.Most desirably, in this embodiment, the essential rep proteins areexpressed under the control of an inducible promoter. In anotherembodiment, the rep proteins are supplied to the host cell in trans.When delivered to the host cell in trans, the rep proteins may bedelivered via a plasmid which contains the sequences necessary to directexpression of the selected rep proteins in the host cell. Mostdesirably, when delivered to the host cell in trans, the plasmidcarrying the capsid protein also carries other sequences required forpackaging the rAAV, e.g., the rep and cap sequences.

Thus, in one embodiment, the rep and cap sequences may be transfectedinto the host cell on a single nucleic acid molecule and exist stably inthe cell as an episome. In another embodiment, the rep and cap sequencesare stably integrated into the chromosome of the cell. Anotherembodiment has the rep and cap sequences transiently expressed in thehost cell. For example, a useful nucleic acid molecule for suchtransfection comprises, from 5′ to 3′, a promoter, an optional spacerinterposed between the promoter and the start site of the rep genesequence, an AAV rep gene sequence, and an AAV cap gene sequence.

Optionally, the rep and/or cap sequences may be supplied on a vectorthat contains other DNA sequences that are to be introduced into thehost cells. For instance, the vector may contain the rAAV constructcomprising the minigene. The vector may comprise one or more of thegenes encoding the helper functions, e.g., the adenoviral proteins E1,E2a, and E4ORF6, and the gene for VAI RNA.

The promoter used in this construct may be any of the constitutive,inducible or native promoters known to one of skill in the art or asdiscussed above. In one embodiment, an AAV P5 promoter sequence isemployed. The selection of the AAV to provide any of these sequencesdoes not limit the invention.

In another embodiment, the promoter for rep is an inducible promoter,such as are discussed above in connection with the transgene regulatoryelements. One preferred promoter for rep expression is the T7 promoter.The vector comprising the rep gene regulated by the T7 promoter and thecap gene, is transfected or transformed into a cell which eitherconstitutively or inducibly expresses the T7 polymerase. SeeInternational Patent Publication No. WO 98/10088, published Mar. 12,1998.

The spacer is an optional element in the design of the vector. Thespacer is a DNA sequence interposed between the promoter and the repgene ATG start site. The spacer may have any desired design; that is, itmay be a random sequence of nucleotides, or alternatively, it may encodea gene product, such as a marker gene. The spacer may contain geneswhich typically incorporate start/stop and polyA sites. The spacer maybe a non-coding DNA sequence from a prokaryote or eukaryote, arepetitive non-coding sequence, a coding sequence withouttranscriptional controls or a coding sequence with transcriptionalcontrols. Two exemplary sources of spacer sequences are the λ phageladder sequences or yeast ladder sequences, which are availablecommercially, e.g., from Gibco or Invitrogen, among others. The spacermay be of any size sufficient to reduce expression of the rep78 andrep68 gene products, leaving the rep52, rep40 and cap gene productsexpressed at normal levels. The length of the spacer may therefore rangefrom about 10 bp to about 10.0 kbp, preferably in the range of about 100bp to about 8.0 kbp. To reduce the possibility of recombination, thespacer is preferably less than 2 kbp in length; however, the inventionis not so limited.

Although the molecule(s) providing rep and cap may exist in the hostcell transiently (i.e., through transfection), it is preferred that oneor both of the rep and cap proteins and the promoter(s) controllingtheir expression be stably expressed in the host cell, e.g., as anepisome or by integration into the chromosome of the host cell. Themethods employed for constructing embodiments of this invention areconventional genetic engineering or recombinant engineering techniquessuch as those described in the references above. While thisspecification provides illustrative examples of specific constructs,using the information provided herein, one of skill in the art mayselect and design other suitable constructs, using a choice of spacers,P5 promoters, and other elements, including at least one translationalstart and stop signal, and the optional addition of polyadenylationsites.

In another embodiment of this invention, the rep or cap protein may beprovided stably by a host cell.

C. The Helper Functions

The packaging host cell also requires helper functions in order topackage the rAAV of the invention. Optionally, these functions aresupplied by a herpesvirus. The necessary helper functions may each beprovided from a human or non-human primate adenovirus source, such asthose described above and/or are available from a variety of sources,including the American Type Culture Collection (ATCC), Manassas, Va.(US). In yet another embodiment, the host cell is provided with and/orcontains an E1a gene product, an E1b gene product, an E2a gene product,and/or an E4 ORF6 gene product. The host cell may contain otheradenoviral genes such as VAI RNA, but these genes are not required. Instill another embodiment, no other adenovirus genes or gene functionsare present in the host cell.

By “adenoviral DNA which expresses the E1a gene product”, it is meantany adenovirus sequence encoding E1a or any functional E1a portion.Adenoviral DNA which expresses the E2a gene product and adenoviral DNAwhich expresses the E4 ORF6 gene products are defined similarly. Alsoincluded are any alleles or other modifications of the adenoviral geneor functional portion thereof. Such modifications may be deliberatelyintroduced by resort to conventional genetic engineering or mutagenictechniques to enhance the adenoviral function in some manner, as well asnaturally occurring allelic variants thereof. Such modifications andmethods for manipulating DNA to achieve these adenovirus gene functionsare known to those of skill in the art.

The adenovirus E1a, E1b, E2a, and/or E4ORF6 gene products, as well asany other desired helper functions, can be provided using any means thatallows their expression in a cell. Each of the sequences encoding theseproducts may be on a separate vector, or one or more genes may be on thesame vector. The vector may be any vector known in the art or disclosedabove, including plasmids, cosmids and viruses. Introduction into thehost cell of the vector may be achieved by any means known in the art oras disclosed above, including transfection, infection, electroporation,liposome delivery, membrane fusion techniques, high velocity DNA-coatedpellets, viral infection and protoplast fusion, among others. One ormore of the adenoviral genes may be stably integrated into the genome ofthe host cell, stably expressed as episomes, or expressed transiently.The gene products may all be expressed transiently, on an episome orstably integrated, or some of the gene products may be expressed stablywhile others are expressed transiently. Furthermore, the promoters foreach of the adenoviral genes may be selected independently from aconstitutive promoter, an inducible promoter or a native adenoviralpromoter. The promoters may be regulated by a specific physiologicalstate of the organism or cell (i.e., by the differentiation state or inreplicating or quiescent cells) or by exogenously added factors, forexample.

D. Host Cells and Packaging Cell Lines

The host cell itself may be selected from any biological organism,including prokaryotic (e.g., bacterial) cells, and eukaryotic cells,including, insect cells, yeast cells and mammalian cells. In oneembodiment, host cells are selected from among any mammalian species,including, without limitation, cells such as A549, WEHI, 3T3, 10T1/2,BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, 293cells (which express functional adenoviral E1), Saos, C2C12, L cells,HT080, HepG2 and primary fibroblast, hepatocyte and myoblast cellsderived from mammals including human, monkey, mouse, rat, rabbit, andhamster. The selection of the mammalian species providing the cells isnot a limitation of this invention; nor is the type of mammalian cell,i.e., fibroblast, hepatocyte, tumor cell, etc. The requirements for thecell used is that it not carry any adenovirus gene other than E1, E2aand/or E4ORF6; it not contain any other virus gene which could result inhomologous recombination of a contaminating virus during the productionof rAAV; and it is capable of infection or transfection of DNA andexpression of the transfected DNA. In a one embodiment, the host cell isone that has rep and cap stably transfected in the cell.

One host cell useful in the present invention is a host cell stablytransformed with the sequences encoding rep and cap, and which istransfected with the adenovirus E1, E2a, and E4ORF6 DNA and a constructcarrying the minigene as described above. Stable rep and/or capexpressing cell lines, such as B-50 (PCT/US98/19463), or those describedin U.S. Pat. No. 5,658,785, may also be similarly employed. Another hostcell contains the minimum adenoviral DNA which is sufficient to expressE4ORF6. Yet other cell lines can be constructed using the novel AAV9 capsequences of the invention.

The preparation of a host cell according to this invention involvestechniques such as assembly of selected DNA sequences. This assembly maybe accomplished utilizing conventional techniques. Such techniquesinclude cDNA and genomic cloning, which are well known and are describedin Sambrook et al., cited above, use of overlapping oligonucleotidesequences of the adenovirus and AAV genomes, combined with polymerasechain reaction, synthetic methods, and any other suitable methods whichprovide the desired nucleotide sequence.

Introduction of the molecules (as plasmids or viruses) into the hostcell may also be accomplished using techniques known to the skilledartisan and as discussed throughout the specification. In preferredembodiment, standard transfection techniques are used, e.g., CaPO₄transfection or electroporation, and/or infection by hybridadenovirus/AAV vectors into cell lines such as the human embryonickidney cell line HEK 293 (a human kidney cell line containing functionaladenovirus E1 genes which provides trans-acting E1 proteins).

Thus, the invention further provides vectors useful in thevaccine/immunization regimens described herein. These, or other AAVvector constructs may be used in regimens for delivery of an immunogenin a regimen involving sequential administration of Ad vectors.

IV. Immunization Regimens

According to the present invention, recombinant vectors are used in theimmunization regimen of the invention for inducing an immune response ina human or non-simian veterinary patient following ex vivo or in vivoadministration. In one embodiment, the immune response induced is ahumoral (i.e., antibody) response to the product expressed by the viralvectors. Depending upon the antigen product expressed, such an antibodyresponse can be specific to the pathogen from which the antigen isderived or cross-reactive with other, related pathogens. In anotherembodiment, the immune response can be a cellular (e.g., CTL) response.Depending upon the immunogenic product expressed, such a CTL responsecan be specific to the pathogen from which the immunogen is derived orcross-reactive with other, related pathogens. In still otherembodiments, both antibody and CTL response may be induced. However, themethod of the invention is advantageous is that it minimizes, and insome cases eliminates, immune response to the viral vector, andparticularly, the adenoviral vector.

Thus, the immunization regimens of the invention can be applied eitherin prophylactic or therapeutic vaccines. Such vaccinal (or otherimmunogenic) compositions are formulated in a suitable delivery vehicle,as described above. Generally, doses for the immunogenic compositionsare in the range defined above for therapeutic compositions. The levelsof immunity of the selected gene can be monitored to determine the need,if any, for boosters. Following an assessment of antibody titers in theserum, optional booster immunizations may be desired.

Optionally, a composition of the invention may be formulated to containviral vectors as described herein, as well as other components,including, e.g. adjuvants, stabilizers, pH adjusters, preservatives andthe like. Suitable exemplary preservatives include chlorobutanol,potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, theparabens, ethyl vanillin, glycerin, phenol, and parachlorophenol.

Suitable chemical stabilizers include gelatin and albumin. Suitableexemplary adjuvants include, among others, immune-stimulating complexes(ISCOMS), LPS analogs including 3-O-deacylated monophosphoryl lipid A(Ribi Immunochem Research, Inc.; Hamilton, Mont.), mineral oil andwater, aluminum hydroxide, Amphigen, Avirdine, L121/squalene, muramylpeptides, and saponins, such as Quil A, and any biologically activefactor, such as cytokine, an interleukin, a chemokine, a ligands, andoptimally combinations thereof. Certain of these biologically activefactors can be expressed in vivo, e.g., via a plasmid or viral vector.For example, such an adjuvant can be administered with a primingadenoviral vector.

The viral vectors used in the invention are administered in “animmunogenic amount”, that is, an amount of virus that is effective in aroute of administration to transfect the desired cells and providesufficient levels of expression of the selected gene to induce an immuneresponse. Where protective immunity is provided, the viruses areconsidered to be vaccine compositions useful in preventing infectionand/or recurrent disease.

Alternatively, or in addition, the vectors used in the invention maycontain nucleic acid sequences encoding a product (e.g., a peptide,polypeptide, or protein) which induces an immune response to a selectedimmunogen. The immunogenic regimen provided herein is expected to behighly efficacious at inducing cytolytic T cells and antibodies to theinserted heterologous antigenic protein expressed by the vector.

Immunogens may be selected from a variety of viral families. Example ofviral families against which an immune response would be desirableinclude, the picornavirus family, which includes the generarhinoviruses, which are responsible for about 50% of cases of the commoncold; the genera enteroviruses, which include polioviruses,coxsackieviruses, echoviruses, and human enteroviruses such as hepatitisA virus; and the genera apthoviruses, which are responsible for foot andmouth diseases, primarily in non-human animals. Within the picornavirusfamily of viruses, target antigens include the VP1, VP2, VP3, VP4, andVPG. Another viral family includes the calcivirus family, whichencompasses the Norwalk group of viruses, which are an importantcausative agent of epidemic gastroenteritis. Still another viral familydesirable for use in targeting antigens for inducing immune responses inhumans and non-human animals is the togavirus family, which includes thegenera alphavirus, which include Sindbis viruses, RossRiver virus, andVenezuelan, Eastern & Western Equine encephalitis, and rubivirus,including Rubella virus. The flaviviridae family includes dengue, yellowfever, Japanese encephalitis, St. Louis encephalitis and tick borneencephalitis viruses.

Other target antigens may be generated from the Hepatitis C [see, e.g.,US Published Patent Application No. US 2003/190606 (Oct. 9, 2003); US2002/081568 (Jun. 27, 2002)] or the coronavirus family, which includes anumber of non-human viruses such as infectious bronchitis virus(poultry), porcine transmissible gastroenteric virus (pig), porcinehemagglutinating encephalomyelitis virus (pig), feline infectiousperitonitis virus (cats), feline enteric coronavirus (cat), caninecoronavirus (dog), and human respiratory coronaviruses, which may causethe common cold and/or non-A, B or C hepatitis. Within the coronavirusfamily, target antigens include the E1 (also called M or matrixprotein), E2 (also called S or Spike protein), E3 (also called HE orhemagglutin-elterose) glycoprotein (not present in all coronaviruses),or N (nucleocapsid). The coronavirus family includes the putativecausative agent for sudden acute respiratory syndrome (SARS). Stillother antigens may be targeted against the rhabdovirus family, whichincludes the genera vesiculovirus (e.g., Vesicular Stomatitis Virus),and the general lyssavirus (e.g., rabies). Within the rhabdovirusfamily, suitable antigens may be derived from the G protein or the Nprotein. The family filoviridae, which includes hemorrhagic feverviruses such as Marburg and Ebola virus, may be a suitable source ofantigens. The paramyxovirus family includes parainfluenza Virus Type 1,parainfluenza Virus Type 3, bovine parainfluenza Virus Type 3,rubulavirus (mumps virus), parainfluenza Virus Type 2, parainfluenzavirus Type 4, Newcastle disease virus (chickens), rinderpest,morbillivirus, which includes measles and canine distemper, andpneumovirus, which includes respiratory syncytial virus (e.g., theglyco-(G) protein and the fusion (F) protein, for which sequences areavailable from GenBank).

The influenza virus is classified within the family orthomyxovirus andis a suitable source of antigen (e.g., the HA protein, the N1 protein).The bunyavirus family includes the genera bunyavirus (Californiaencephalitis, La Crosse), phlebovirus (Rift Valley Fever), hantavirus(puremala is a hemahagin fever virus), nairovirus (Nairobi sheepdisease) and various unassigned bungaviruses. The arenavirus familyprovides a source of antigens against LCM and Lassa fever virus. Thereovirus family includes the genera reovirus, rotavirus (which causesacute gastroenteritis in children), orbiviruses, and cultivirus(Colorado Tick fever, Lebombo (humans), equine encephalosis, bluetongue).

The retrovirus family includes the sub-family oncorivirinal whichencompasses such human and veterinary diseases as feline leukemia virus,HTLVI and HTLVII, lentivirinal (which includes human immunodeficiencyvirus (HIV), simian immunodeficiency virus (SIV), felineimmunodeficiency virus (FIV), equine infectious anemia virus, andspumavirinal). Among the lentiviruses, many suitable antigens have beendescribed and can readily be selected. Examples of suitable HIV and SIVantigens include, without limitation the gag, pol, Vif, Vpx, VPR, Env,Tat, Nef, and Rev proteins, as well as various fragments thereof. Forexample, suitable fragments of the Env protein may include any of itssubunits such as the gp120, gp160, gp140, gp41, or smaller fragmentsthereof, e.g., of at least about 8 amino acids in length. Similarly,fragments of the tat protein may be selected. [See, U.S. Pat. No.5,891,994 and U.S. Pat. No. 6,193,981.] See, also, the HIV and SIVproteins described in D. H. Barouch et al, J. Virol., 75(5):2462-2467(March 2001), and R. R. Amara, et al, Science, 292:69-74 (6 Apr. 2001).In another example, the HIV and/or SIV immunogenic proteins or peptidesmay be used to form fusion proteins or other immunogenic molecules. See,e.g., the HIV-1 Tat and/or Nef fusion proteins and immunization regimensdescribed in WO 01/54719, published Aug. 2, 2001, and WO 99/16884,published Apr. 8, 1999. The invention is not limited to the HIV and/orSIV immunogenic proteins or peptides described herein. In addition, avariety of modifications to these proteins has been described or couldreadily be made by one of skill in the art. See, e.g., the modified gagprotein that is described in U.S. Pat. No. 5,972,596. Further, anydesired HIV and/or SIV immunogens may be delivered alone or incombination. Such combinations may include expression from a singlevector or from multiple vectors. Optionally, another combination mayinvolve delivery of one or more expressed immunogens with delivery ofone or more of the immunogens in protein form. Additional sources oftarget antigens and immunogens are discussed in more detail below.

The papovavirus family includes the sub-family polyomaviruses (BKU andJCU viruses) and the sub-family papillomavirus (associated with cancersor malignant progression of papilloma). Examples of papillomavirusproteins useful as immunogenic products include those derived from thepapilloma virus “early” and “late” genes designated E1 to E7, L1 and L2.See, e.g., US Published Patent Application No. 2002/0137720 [Ertl].Other papillomavirus antigens and combinations thereof have beendescribed. See, e.g., US Published Application No. 2003/129199 (Jul. 10,2003); US Published Application No. 2002/18221 (Dec. 15, 2002); U.S.Pat. No. 6,342,224.

The adenovirus family includes viruses (EX, AD7, ARD, O.B.) which causerespiratory disease and/or enteritis. The parvovirus family felineparvovirus (feline enteritis), feline panleucopeniavirus, canineparvovirus, and porcine parvovirus. The herpesvirus family includes thesub-family alphaherpesvirinae, which encompasses the genera simplexvirus(HSVI, HSVII), varicellovirus (pseudorabies, varicella zoster) and thesub-family betaherpesvirinae, which includes the genera cytomegalovirus(Human CMV), muromegalovirus) and the sub-family gammaherpesvirinae,which includes the genera lymphocryptovirus, EBV (Burkitts lymphoma),infectious rhinotracheitis, Marek's disease virus, and rhadinovirus. Thepoxvirus family includes the sub-family chordopoxyirinae, whichencompasses the genera orthopoxvirus (Variola (Smallpox) and Vaccinia(Cowpox)), parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus,suipoxvirus, and the sub-family entomopoxyirinae. The hepadnavirusfamily includes the Hepatitis B virus. One unclassified virus which maybe suitable source of antigens is the Hepatitis delta virus. Still otherviral sources may include avian infectious bursal disease virus andporcine respiratory and reproductive syndrome virus. The alphavirusfamily includes equine arteritis virus and various Encephalitis viruses.

The present invention may also encompass regimens utilizing productwhich are useful to immunize a human or non-human animal against otherpathogens including bacteria, fungi, parasitic microorganisms ormulticellular parasites which infect human and non-human vertebrates, orfrom a cancer cell or tumor cell. Examples of bacterial pathogensinclude pathogenic gram-positive cocci include pneumococci;staphylococci; and streptococci. Pathogenic gram-negative cocci includemeningococcus; gonococcus. Pathogenic enteric gram-negative bacilliinclude enterobacteriaceae; pseudomonas, acinetobacteria and eikenella;melioidosis; salmonella; shigella; haemophilus (Haemophilus influenzae,Haemophilus somnus); moraxella; H. ducreyi (which causes chancroid);brucella; Franisella tularensis (which causes tularemia); yersinia(pasteurella); streptobacillus moniliformis and spirillum. Gram-positivebacilli include listeria monocytogenes; erysipelothrix rhusiopathiae;Corynebacierium diphtheria (diphtheria); cholera; B. anthracis(anthrax); donovanosis (granuloma inguinale); and bartonellosis.Diseases caused by pathogenic anaerobic bacteria include tetanus;botulism; other clostridia; tuberculosis; leprosy; and othermycobacteria.

Examples of specific bacterium species are, without limitation,Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcusagalactiae, Streptococcus faecalis, Moraxella catarrhalis, Helicobacterpylori, Neisseria meningitidis, Neisseria gonorrhoeae, Chlamydiatrachomatis, Chlamydia pneumoniae, Chlamydia psittaci, Bordetellapertussis, Salmonella typhi, Salmonella typhimurium, Salmonellacholeraesuis, Escherichia coli, Shigella, Vibrio cholerae,Corynebacterium diphtheriae, Mycobacterium tuberculosis, Mycobacteriumavium, Mycobacterium intracellulare complex, Proteus mirabilis, Proteusvulgaris, Staphylococcus aureus, Clostridium tetani, Leptospirainterrogans, Borrelia burgdorferi, Pasteurella haemolytica, Pasteurellamultocida, Actinobacillus pleuropneumoniae and Mycoplasma gallisepticum.

Pathogenic spirochetal diseases include syphilis; treponematoses: yaws,pinta and endemic syphilis; and leptospirosis. Other infections causedby higher pathogen bacteria and pathogenic fungi include actinomycosis;nocardiosis; cryptococcosis (Cryptococcus), blastomycosis (Blastomyces),histoplasmosis (Histoplasma) and coccidioidomycosis (Coccidiodes);candidiasis (Candida), aspergillosis (Aspergillis), and mucormycosis;sporotrichosis; paracoccidiodomycosis, petriellidiosis, torulopsosis,mycetoma and chromomycosis; and dermatophytosis. Rickettsial infectionsinclude Typhus fever, Rocky Mountain spotted fever, Q fever, andRickettsialpox. Examples of mycoplasma and chlamydial infectionsinclude: mycoplasma pneumoniae; lymphogranuloma venereum; psittacosis;and perinatal chlamydial infections. Pathogenic eukaryotes encompasspathogenic protozoans and helminths and infections produced therebyinclude: amebiasis; malaria; leishmaniasis (e.g., caused by Leishmaniamajor); trypanosomiasis; toxoplasmosis (e.g., caused by Toxoplasmagondii); Pneumocystis carinii; Trichans; Toxoplasma gondii; babesiosis;giardiasis (e.g., caused by Giardia); trichinosis (e.g., caused byTrichomonas); filariasis; schistosomiasis (e.g., caused by Schistosoma);nematodes; trematodes or flukes; and cestode (tapeworm) infections.Other parasitic infections may be caused by Ascaris, Trichuris,Cryptosporidium, and Pneumocystis carinii, among others.

Many of these organisms and/or toxins produced thereby have beenidentified by the Centers for Disease Control [(CDC), Department ofHeath and Human Services, USA], as agents which have potential for usein biological attacks. For example, some of these biological agents,include, Bacillus anthracis (anthrax), Clostridium botulinum and itstoxin (botulism), Yersinia pestis (plague), variola major (smallpox),Francisella tularensis (tularemia), and viral hemorrhagic fevers[filoviruses (e.g., Ebola, Marburg], and arenaviruses [e.g., Lassa,Machupo]), all of which are currently classified as Category A agents;Coxiella burnetti (Q fever); Brucella species (brucellosis),Burkholderia mallei (glanders), Burkholderia pseudomallei (meloidosis),Ricinus communis and its toxin (ricin toxin), Clostridium perfringensand its toxin (epsilon toxin), Staphylococcus species and their toxins(enterotoxin B), Chlamydia psittaci (psittacosis), water safety threats(e.g., Vibrio cholerae, Crytosporidium parvum), Typhus fever (Richettsiapowazekii), and viral encephalitis (alphaviruses, e.g., Venezuelanequine encephalitis; eastern equine encephalitis; western equineencephalitis); all of which are currently classified as Category Bagents; and Nipan virus and hantaviruses, which are currently classifiedas Category C agents. In addition, other organisms, which are soclassified or differently classified, may be identified and/or used forsuch a purpose in the future. It will be readily understood that theviral vectors and other constructs described herein are useful todeliver antigens from these organisms, viruses, their toxins or otherby-products, which will prevent and/or treat infection or other adversereactions with these biological agents.

Administration of the vectors according to the invention to deliverimmunogens against the variable region of the T cells elicit an immuneresponse including CTLs to eliminate those T cells. In rheumatoidarthritis (RA), several specific variable regions of T-cell receptors(TCRs) which are involved in the disease have been characterized. TheseTCRs include V-3, V-14, V-17 and Vα-17. Thus, delivery of a nucleic acidsequence that encodes at least one of these polypeptides will elicit animmune response that will target T cells involved in RA. In multiplesclerosis (MS), several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs include V-7and Vα-10. Thus, delivery of a nucleic acid sequence that encodes atleast one of these polypeptides will elicit an immune response that willtarget T cells involved in MS. In scleroderma, several specific variableregions of TCRs which are involved in the disease have beencharacterized. These TCRs include V-6, V-8, V-14 and Vα-16, Vα-3C, Vα-7,Vα-14, Vα-15, Vα-16, Vα-28 and Vα-12. Thus, delivery of a recombinantsimian adenovirus that encodes at least one of these polypeptides willelicit an immune response that will target T cells involved inscleroderma.

Further, desirable immunogens include those directed to eliciting atherapeutic or prophylactic anti-cancer effect in a vertebrate host,such as, without limitation, those utilizing a cancer antigen ortumor-associated antigen including, without limitation, prostatespecific antigen, carcino-embryonic antigen, MUC-1, Her2, CA-125 andMAGE-3.

Suitably, the adenoviral vectors and AAV vectors are delivered in acombination regimen involving sequential administration, orcoadministration, of an adenoviral vector and an AAV vector. Theseregimens can further include sequential or coadministrationadministration with one or more additional adenovirus vectors, e.g., afunctionally E1-deleted, functionally E4-deleted adenovirus, one or moreadditional AAV vectors, or other therapeutic and/or vaccine agents.

In one embodiment, the regimen further involves administration of a DNAvaccine, e.g., via gene gun or plasmid. Such a DNA vaccine may be usedas a priming step, which precedes adenoviral-mediated and/orAAV-mediated delivery according to the invention. Alternatively, such aDNA vaccine may be used as a boost following one or moreadenoviral-mediated and/or AAV-mediated delivery of the invention.

In another embodiment, the regimen further involves sequential orco-administration of a protein-based vaccine. Such a vaccine can be usedas a boost, following adenoviral-mediated and/or AAV-mediated deliveryaccording to the invention. Alternatively, such a protein-based vaccinemay be used as a prime, or in between one or more adenoviral-mediatedand/or AAV-mediated immunizations in a regimen of the invention.

In one example, an immunization regimen of the invention provides aprotective immune response to the virus, bacteria or other organism,from which the antigen is derived, or a cross-reactive virus, bacteriaor other source. In another example, the immunization regimen describedherein can include a multiprotein regimen. See, e.g., R. R. Amara,Science, 292:69-74 (6 Apr. 2001) which describes a multiprotein regimenfor expression of protein subunits useful for generating an immuneresponse against HIV and SIV.

In another desired embodiment, the regimen provides a therapeutic immuneeffect. Optionally, these effects can be measured using assays fordetection of the presence of antigen-specific antibodies, T cells, orthe condition for which therapy is being administered.

The vectors used in an immunization regimen of the invention can beadministered at various sites in the body in a dose dependent manner,which depends on the product to which the desired immune response isbeing targeted. The invention is not limited to the amount or situs ofinjection(s) or to the pharmaceutical carrier. Rather, the regimen mayinvolve a priming and boosting step, each of which may include a singledose or dosage that is administered hourly, daily, weekly or monthly, oryearly. The amount or site of delivery is desirably selected based uponthe identity and condition of the mammal.

The dosage unit of the vector suitable for delivery of the antigen tothe mammal is described herein. The vector is prepared foradministration by being suspended or dissolved in a pharmaceutically orphysiologically acceptable carrier such as isotonic saline; isotonicsalts solution or other formulations that will be apparent to thoseskilled in such administration. The appropriate carrier will be evidentto those skilled in the art and will depend in large part upon the routeof administration. The compositions used in the invention areadministered to a mammal according to the routes described above, in asustained release formulation using a biodegradable biocompatiblepolymer, or by on-site delivery using micelles, gels and liposomes.Optionally, the priming step of this invention also includesadministering with the priming composition, a suitable amount of anadjuvant, such as are defined herein.

Dosages of the viral vector will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among mammalian (including human) patients.Advantageously, the unexpected potency of the recombinant simian (e.g.,chimpanzee) adenoviruses of the invention permits the use significantlylower amount of the recombinant chimpanzee adenovirus to provide aneffective amount to induce the desired immunogenic effect (e.g.,induction of a predetermined level of antibodies and/or CD8+ T cells).

For example, for small animals, an effective dose of an adenoviralvector may be provided by 10⁵ particles/animal and 10¹¹ particles/animalof adenovirus. For a larger animal, e.g., about 80 kg, 10⁷ to about 10¹³particles per subject may be useful. However, higher doses may bereadily selected, e.g., depending upon the selected route of delivery.For example, the adenoviral vector may be delivered in an amount whichranges from about 100 μL to about 100 ml, and more preferably, about 1mL to about 10 mL, of carrier solution.

In one embodiment, an effective dosage of the AAV vector is generally inthe range of from about 0.1 ml to about 100 ml of solution containingconcentrations of from about 1×10⁹ to 1×10¹⁶ genomes virus vector. Apreferred human dosage for delivery to large organs (e.g., liver,muscle, heart and lung) may be about 5×10¹⁰ to 5×10¹³ AAV genomes per 1kg, at a volume of about 1 to 100 mL. A preferred dosage for delivery toeye is about 5×10⁹ to 5×10¹² genome copies, at a volume of about 0.1 mLto 1 mL.

Depending upon the desired routes of administration, one of skill in theart can select an appropriate regimen. In general, a second, orsubsequent immunization, composition can be administered about 2 toabout 27 weeks after administering the preceding immunizationcomposition, to the mammalian subject. The administration of thesubsequent composition is accomplished using an effective amount of acomposition containing or capable of delivering the same antigen asadministered by the prior composition. Desirably, the product of theboosting composition is the same, or cross-reactive, as that encoded bythe priming composition.

The time period between sequential administrations, according to thepresent invention, can be adjusted according to the order of Ad-mediateddelivery, AAV-mediated delivery, and any optional additional priming orboosting compositions (e.g., DNA-based or protein-based vaccines). Forexample, peak immune response is generally observed about 10 to 14 daysfollowing an Ad-mediated delivery. However, boosting following this peakmay generate a second peak. Thus, it may be desirable to time expressionof a boosting antigen to express from about 10 to 21 days, or 18 to 28days, or 28 days to 27 weeks following Ad-mediated delivery. In anotherexample, certain AAV serotypes demonstrate peak expression about 3 to 4weeks following delivery. Thus, boosting following AAV delivery may betimed to express the antigen, or a cross-reactive antigen, about 3 weeksto about 4 weeks, about 4 weeks to about 2 months, or about 2 months to27 weeks months, or longer, following AAV delivery.

In one embodiment, the heterologous expression cassette delivered via aviral vector, e.g., an AAV vector, according to the invention containsthe immunogenic product operably linked to an inducible or regulatablepromoter. When used in a regimen according to the present invention, theinducing or regulating agent is typically administered such thatexpression of product is activated immediately upon administration ofthe viral vector. Thereafter, expression can be extinguished by removalof the inducing or regulating agent. The regimen of the invention caninclude “pulse” activation of expression. Thus, the method of theinvention permits expression to be induced, extinguished, and then againinduced after a period of time. In another embodiment, expression isinduced not upon administration, but several days to several weeksfollowing administration. This embodiment may permit co-delivery of anAd and AAV followed by induction (activation) several weeks followingdelivery, depending upon the delay caused by the induction agent. Forexample, once an inducing agent is delivered, it may be 7 to 10 days orlonger before the effect is observed. One of skill in the art will befamiliar with the delay between delivery of the inducing or activatingagent and the effect and will be able to readily factor this into theselected regimen.

The therapeutic levels or levels of immunity, of the selected gene canbe monitored to determine the need, if any, for boosters. Following anassessment of CD8+ T cell response, or, antibody titers, in the serum,optional additional booster immunizations may be desired.

In another aspect, the invention provides a product useful forperforming the immunization regimens described herein.

Such a product can contain one or more of the adenoviral vectorsdescribed herein in a suitable container. Typically, such a product willfurther contain instructions for administration of the adenoviralvectors.

Further, the product may contain a physiologically acceptable carriersuitable for the selected route of delivery, e.g., for dilution and/orreconstitution of one or more the adenoviral vectors, syringes, vials,and the like.

EXAMPLES

The following examples are provided to illustrate the invention and donot limit the scope thereof. One skilled in the art will appreciate thatalthough specific reagents and conditions are outlined in the followingexamples, modifications can be made that are meant to be encompassed bythe spirit and scope of the invention.

Recently, a strategy involving sequential immunizations withheterologous vaccine carriers expressing the same antigen has resultedin the generation of unparalleled levels of specific immunity and, insome cases, afforded protection against infectious agents.

Replication-defective, adenoviral vectors based on human serotype 5 (H5)can induce robust cellular and humoral immune responses against thetransgene product. In addition, adeno-associated viral (AAV) vectorshave also been shown to elicit antigen-specific immune responses.

Example 1

For the studies described in Example 2, Ebola Zaire virus envelopeglycoprotein (Ebo GP) was used as a model antigen to create H5CMVGP andAAV-1CMVGP vaccine vectors. AAV serotype I was chosen because it ishighly efficient in transducing muscle.

The vectors used in these studies were generated using conventionalmethods, as follows.

A. Generation of Adenoviral Vector

1. Creation of Molecular Clones of EboZ Expressing Adenovirus Vectors

Recombinant adenovirus genomes that derived from different species andstrains of adenoviruses and express EboZGP were created through directligation and green/white selection system that was described elsewhere[Gao et al., Gene Therapy, 2003 and Roy et al., Human Gene Therapy,15(5):519-530 (May 2004)]. Briefly, the EboZGP cDNA was subcloned into auniversal pShuttle plasmid vector between CMV promoter and bovine growthhormone poly A which was used for introducing the EboZGP into a varietyof molecular clones of adenovirus backbones.

The molecular clones of adenovirus backbones include Human serotype 5with E1 and E3 deletions (H5.040), Chimpanzee serotype 7 with E1deletion only (C7.000), E1 and E3 deletions (C7.010) and E1 and E4deletions (C7.001). The cloning process to create those molecular cloneswere described elsewhere [Gao et al., Gene Therapy, cited above, 2003and Roy et al., Human Gene Therapy, 15(5):519-530 (May 2004)]. All thesemolecular clones containing a cassette that expressed prokaryotic GFPfrom bacterial lac promoter and flanked by two rare restriction sites,PI-Sce I and I-Ceu I. This allowed the EboZ expression cassette from theuniversal pShuttle construct to be swapped into the adenovirus molecularclones through a convenient and efficient green/white selection mediatedcloning process (Gao et al., Gene Therapy, 2003).

2. Rescue, Expansion and Purification of AdEboZ Vectors

To rescue recombinant viruses from the molecular clones, the plasmidDNAs were linearized by appropriate restriction enzymes to release thevector genomes from plasmid backbones and transfected into appropriatecell lines. For E1/E4 deleted vectors, 10-3 cells, a 293 cell basedE1/E4-complementing cell line with E4ORF6 expressed under Zinc inductionwere used. For all other constructs, 293 cells were used.

Once full cytopathic effect (CPE), the sign of virus rescue andreplication, was observed, crude viral lysate harvested for gradualexpansion to large scale infections in appropriate cell lines. Viruseswere purified by the standard CsCl gradient sedimentation method. Thegenome structures of recombinant viruses were confirmed by restrictionenzyme analysis. For all vector except for E1/E4-deleted vectors,infectivity of the viruses were determined by plaque assay on 293 cells.However, the vectors used for immunization experiments were dosed basedon virus physical particle numbers measured by OD₂₆₀ readings on aUV-spectrophometer.

B. Creation and Production of MV2/1CMVEboZGP Vector

To create the recombinant AAV2 genome expressing EboZ glycoprotein, theEGFP insert in pAAV2CMVEGFP plasmid [E. M. Surace et al, J. Virol.,77(14):7957-7963 (July 2003) was replaced with the EboZGP cDNA.

Recombinant AAV2/1CMVEboZGP virus was produced by the tripletransfection method in 293 cells to transencapsidate the AAV2CMVEboZGPgenome with AAV1 capsids. The vector was purified by CsCl sedimentationmethod. The genome copy titer of the vector was determined by real timePCR, whereas its infectious titer was analyzed by the infectious centerassay. The vector doses for vaccination were based on the genome titer.

Example 2 Immunization Regimen of Invention Boosts Immune Response toAntigen

B10BR mice (6-8 weeks old) were purchased from The Jackson Laboratory(Bar Harbor, Me.) and kept at the Animal Facility of The WistarInstitute (Philadelphia, Pa.). Mice were immunized with recombinantadenoviral vectors or recombinant adeno-associated viral vectors dilutedin 100 μl PBS at the doses provided below by intramuscular injection.More particularly, priming was with recombinant adenoviral vectors wasat 5×10¹⁰, 5×10⁹, or 5×10⁸ particles/mouse of H5GP vector. Boosting waswith 5×10¹⁰ genome copies/mouse of AAV2/1 GP vector. Serum from 3 micein each group were collected and pooled at either day 42 after injection(pre-boost), or 1 week after boost, or 2 weeks after boost, as indicatedin the FIGURE. Total IgG responses to GP were measured by ELISA.

A. Antigen-Specific Immune Response

GP-specific T cell and B cell responses elicited in B10BR mice wereanalyzed after vaccination by IM injection with either H5CMVGP orAAV-1CMVGP vectors alone.

The TELRTFSI [SEQ ID NO: I] peptide which carries the immunodominant MHCclass I epitope of EboZ GP for mice of the H-2^(k) haplotype wassynthesized by Mimotopes (Victoria, Australia). Peptide was diluted inDMSO to a concentration of 5 mg/ml and stored at −80° C. Peptide wasused at 2 μg/ml and DMSO concentrations were kept below 0.1% (v/v) inall final assay mixtures.

Splenocytes from immunized mice were stimulated with H-2^(K) restrictedEboZ GP-specific peptide (TELRTFSI, SEQ ID NO:1) for 5 hours at 37° C.and 10% CO₂ in the presence of 1 μl/ml Brefeldin A (GolgiPlug, BDPharMingen, San Diego, Calif.). Control cells were incubated withoutpeptide. After washing, cells were stained with a FITC-labeledanti-mouse CD8 antibody (BD PharMingen). Then, cells will be washed andpermeabilized in Cytofix/Cytoperm (BD PharMingen) for 20 minutes on ice.Subsequently, cells were washed again and stained with a PE-labeledanti-mouse IFN-γ antibody (BD PharMingen). After extensively washing,cells were examined by two-color flow cytometry and data were analyzedby WinMDi software. Splenoctyes incubated without the peptide to GPshowed<0.5% IFN-gamma producing CD8+ T cells.

AAV-1CMVGP vectors at a dose of 5×10¹⁰ genome copies per mouse inducedmuch lower frequencies of CD8⁺ T cell producing IFN-γ by intracellularcytokine staining with H-2^(k) restricted GP-specific peptide asstimulant and less total IgG response to GP measured by ELISA thanH5CMVGP at a dose of 5×10⁸ particles per mouse.

B. Enhanced Immune Response to Antigen

Immune responses elicited in B10BR mice were examined after priming withH5CMVGP (5×10⁸ particles/mouse) and boosting with AAV-1CMVGP (5×10⁹genome copies/mouse).

Mice were bled either by retro-orbital puncture at various times afterimmunization or by heart-puncture at the termination. Sera were preparedand tested for total IgG response to EboZ GP on 96-well plates coatedwith EboZ VLPs diluted in PBS. The plates were coated overnight at 4° C.and blocked for 2 hours with PBS containing 3% bovine serum albumin(BSA) at room temperature. After washing, sera diluted in PBS containing1% BSA were added onto wells for 2 hours at room temperature. Afterwashing, a 1:10,000 dilution of horseradish peroxidase-conjugated goatanti-mouse IgG (Sigma Chemicals, St. Louis, Mo.) was added to the wellsfor 1 hour at room temperature. After washing, TMB substrate (SigmaChemicals) was added for 10-20 minutes and reaction was subsequentlystopped by adding Stop Reagent (Sigma Chemicals). Optical density wasred at 450 nm. A cut-off value for positive sample was calculated as themean delta OD at 450 nm for naïve serum at a 1:100 dilution plus 3 timesof standard deviations. The endpoint antibody titer of each sampletested was then defined as the reciprocal of the highest dilution of theserum with a delta OD at 450 nm, which was interpolated according to thelinear regression analysis, above the cut-off value.

A significant increase of total IgO response against GP was observed asearly as 10 days after boost. More important, the total IgG responseagainst GP achieved by this prime/boost strategy was at least 2 to 4fold higher than that generated by H5CMVGP alone even at a dose of5×10¹⁰ particles per mouse. See, FIG. 1.

Example 3 Immunization Against Ebola Using Capsid Modified Versions ofAAV8 and Adenovirus Expressing Ebola Envelope Glycoproteins

A. Generation of MV 8 with Insertion Cassettes

Modified vp1 proteins having inserts at amino acid residue position 587of AAV8 are being generated using a first class of peptides designed toincrease dendritic cell (DC) transduction by the incorporation of knownDC binding motifs. These peptides have been described [T. J. Curiel, etal., J. Immunol., 172(12):7423-31 (2004)].

In order to make AAV more attractive as a vaccine carrier, we willattempt to enhance the ability of the capsid to activate innateimmunity. A panel of selected molecules that have been described toenhance cellular immune responses, some of them in a known TLR dependentmanner, will be fused to the AAV capsid. In this rationale the capsidwill generate the proper inflammatory context for recruitment andactivation of APCs. While the engineered capsid acts as a danger signal,the transgene expression will then act to prime the adaptive immunesystem.

Protein/ Target Class peptide #AA cells I DC3 12 DC I DC12 12 DC I DC1812 DC

Insertion in position 587 will be accomplished by insertionalmutagenesis making use of splicing by overlap extension [Horton, R. M.,et al., Gene, 1989, 77(10):61-68]. Two sets of primers will be designedthat give rise to two fragments flanking the 587 insertion site. Theinsertion itself will be encoded on the 5′ tail of the internal primersmaking sure that there is sufficient overlap for annealing in thesplicing step. The external 2 primers will encode for the conservedBsiWI and EcoRV sites in the packaging plasmids p5E18 (AAV2), pAAV2/1and pAAV2/8. In a second PCR reaction those two fragments will bespliced together in the presence of the two external primers and the twofragments. The spliced fragment will then be digested with BsiWI andEcoRV and subsequently ligated to the similarly digested paternalpackaging vector to generate the trans plasmid pAAV2/8 (modified).

The AAV2/8 vector having the modified capsid is then generated usingknown methods. See, e.g., Example 1.

B. Generation of MV8 with VP2 Fusion Proteins

Fusion proteins using the class 1 molecules DC3, DC12 and DC18 will begenerated using the methods described herein.

In addition, vp2 fusion proteins are being generated using a secondclass of molecules aimed at engaging the innate immune system throughknown receptor-ligand interaction. This process is expected to yield thepro-inflammatory milieu essential for a productive adaptive immuneresponse. Whereas a strong pro-inflammatory response is undesirable forgene therapy, it is considered advantageous for vaccine purposes.

Protein/ Peptide Common Name Organism #AA Ligand Target Cells FlaAFlagellin L. 287 TLR5 Mono monocytogenes DC Hsp60 Heat Shock Human 573TLR4 Mono Protein DC C3d Complement Human/Mouse 294 CD21 B cellComponent 3 (CR2) fDCs Fragment d #AA: number of Amino Acids, RGD motif:a tripeptide motif Arg-Gly- Differentiation, (f)DC: (follicular)Dendritic Cell, Mono: Monocytes Asp supports cell adhesion throughbinding a subset of integrin molecules. TLR: Toll Like Receptor, CD:Cluster of differentiation.

The FlaA protein has been described [Hayashi, F., et al., Nature, 2001.410(6832): p. 1099-103]. The Hsp60 protein has been described [Ohashi,K., et al., J Immunol, 2000. 164(2): p. 558-61]; the C3d protein hasbeen described [D'Souza, S. E., et al., Trends Biochem Sci, 1991. 16(7):p. 246-50; M. C. Carroll, Annu Rev Immunol, 1998. 16: p. 545-68].

For the VP2 N-terminal fusion, a similar set of packaging plasmidsdescribed by Warrington et al., cited above, will be made for AAV8. AVP1,3 only expressing vector and a plasmid expressing VP2 from a mutatedstrong start ATG will be constructed as described by the authors. Inshort, for the VP1,3 expressing plasmid the VP2 start codon will bemutated by site directed mutagenesis into GCG coding for Alanine. TheVP2 strong start plasmid (VP2A) will be obtained by subsequentlymutating the VP1 and VP3 ATG into CTG followed by the introduction ofATG as the VP2 start.

In order to obtain a flexible system for insertions into position 138, alinker was introduced in the VP2A packaging plasmids. Making use of theQuickchange™ mutagenesis kit (Stratagene, La Jolla, Calif.) we willinsert the unique NheI and ClaI sites right after the start codon of VP2at AA position 138. All peptides can then be inserted by PCR from thecDNA making use of flanking primers encoding for the NheI (5′) and ClaI(3′) sites. In this way the peptides can be introduced in the differentVP2A packaging plasmids in a uniform way.

The AAV2/8 vector having the modified capsid is then generated usingthese trans plasmids and known methods. See, e.g., Example 1.

C. Immunization Regimen with (A)

Animals are primed intramuscularly with an adenoviral vector, e.g.,AdH5EboZ GP, at different doses. Six weeks later, animals are boostedwith a modified AAV8 (having a vp1 insertion cassette, prepared asdescribed in A) carrying EboZ GP at a dose of 5×10¹⁰ genome copies peranimal. GP-specific CD8⁺ T-cell response and total IgG titer can bemeasured both before and after boost.

A significant increase in a GP-specific cellular immune response and/oran improved humor immune response.

D. Immunization Regimen with (B)

Animals are primed intramuscularly with an adenoviral vector, e.g.,AdH5EboZ GP, at different doses. Six weeks later, animals are boostedwith the modified AAV8 (having a vp2 fusion protein, prepared asdescribed in step A) carrying the same antigen (EboZ GP) at a dose of5×10¹⁰ genome copies per animal. GP-specific CD8⁺ T-cell response andtotal IgG titer can be measured both before and after boost.

A significant increase in GP-specific IgG titer by ELISA after boost isanticipated.

For the FlaA-containing modified AAV8 capsid, improved DC activation andimproved Ag expressing transgene introduced into the antigen-presentingcell is anticipated.

For the hsp60 and C3d-containing modified AAV8 capsids, activation ofinnate immunity is anticipated.

All publications cited in this specification, and the sequence listing,are incorporated herein by reference. While the invention has beendescribed with reference to particular embodiments, it will beappreciated that modifications can be made without departing from thespirit of the invention. Such modifications are intended to fall withinthe scope of the appended claims.

1. An immunization regimen comprising delivering to a subject: (a) areplication-defective adenovirus comprising a first heterologousexpression cassette comprising a nucleic acid sequence encoding aproduct for inducing an immune response to said product under thecontrol of regulatory control sequences which direct expression of saidproduct; and (b) an adeno-associated virus (AAV) comprising a secondheterologous expression cassette comprising a nucleic acid sequenceencoding said product for inducing an immune response to said productunder the control of regulatory control sequences which directexpression of said product, wherein the AAV is delivered prior todelivery of the replication-defective adenovirus.
 2. The regimenaccording to claim 1, wherein the regulatory sequences in the first andsecond expression cassettes of the replication-defective adenovirus andadeno-associated virus differ.
 3. The regimen according to claim 1,wherein the regulatory sequences of the adeno-associated virus comprisean inducible promoter.
 4. The regimen according to claim 3, furthercomprising delivering an inducing agent to the subject followingadministration of the AAV.
 5. The regimen according to claim 4, whereinthe inducing agent is administered to the subject followingadministration of the adenovirus.
 6. The regimen according to claim 1,wherein the adenovirus further comprises a deletion in the regionselected from the group of adenovirus regions consisting of E2, E3, E4,L1, L2, L3, L4, and L5 region.
 7. The regimen according to claim 1,wherein the expression cassette of the adenovirus is located in site ofan E1 region.
 8. The regimen according to claim 1, wherein the firstexpression cassette of the adenovirus and/or the second expressioncassette of the adeno-associated virus is located in an deleted E3region.
 9. The regimen according to claim 1, wherein the adenovirus isof a serotype selected from the group consisting of C5, C6, C7, C68 andC1.
 10. The regimen according to claim 1, wherein the adenovirus isadministered orally, intranasally, or intramuscularly.
 11. The regimenaccording to claim 1, wherein the AAV is of a serotype selected from thegroup consisting of AAV1 and AAV8.
 12. The regimen according to claim 1,wherein the AAV is administered intramuscularly.
 13. The regimenaccording to claim 1, wherein the regimen further comprisesadministering a DNA vaccine.
 14. The regimen according to claim 13,wherein said DNA vaccine is administered prior to the AAV vector. 15.The regimen according to claim 1, further comprising administering aprotein-based vaccine.
 16. The regimen according to claim 15, whereinsaid protein-based vaccine is delivered after the adenoviral vector. 17.The regimen according to claim 15, wherein said protein-based vaccine isdelivered after the AAV vector.
 18. The regimen according to claim 1,wherein the AAV vector has a modified capsid comprising a heterologouspeptide or polypeptide that enhances antigenicity of the AAV vector. 19.The regimen according to claim 18, wherein the modified capsid comprisesa vp2 protein having a peptide fused to its N-terminus.
 20. The regimenaccording to claim 18, wherein the modified capsid comprises a modifiedvp1 protein having a peptide inserted therein.
 21. A method of inducingan antigen-specific humoral or T-cell response in a subject, comprisingadministering: (a) a replication-defective adenovirus vector comprisinga first heterologous expression cassette comprising a nucleic acidsequence encoding a product for inducing an antigen-specific humoral orT-cell response to said product under the control of regulatory controlsequences which direct expression of said product; and (b) anadeno-associated virus (AAV) vector comprising a second heterologousexpression cassette comprising nucleic acid sequence encoding saidproduct for inducing an antigen-specific humoral or T-cell response tosaid product under the control of regulatory control sequences whichdirect expression of said product wherein said replication-defectiveadenovirus vector is administered after said adeno-associated virus(AAV) vector.
 22. The method according to claim 21, further comprisingadministration of multiple adeno-associated virus (AAV) vectors beforeadministration of said replication-defective adenovirus vector.
 23. Themethod according to claim 21, further comprising administration ofmultiple replication-defective adenovirus vectors after saidadeno-associated virus (AAV) vector.